Development and validation of a real-time PCR for the molecular identification of Thaumatotibia leucotreta (Meyrick, 1913) (Lepidoptera: Tortricidae: Olethreutinae) intercepted in trade

Abstract

The false codling moth, Thaumatotibia leucotreta (Meyrick), is a polyphagous pest indigenous to most of sub-Saharan Africa. This species is considered to be quarantine throughout much of the world, in part due to its extensive host range and significant damage caused by larval feeding. We developed a specific real-time PCR assay that allows for rapid and reliable identification of T. leucotreta. More than 150 target specimens were sequenced using an Illumina whole-genome shotgun approach to identify the most suitable loci for assay development. A hydrolysis probe that binds to a segment of the internal transcribed spacer 2 (ITS2) region was designed and used with a generic internal control targeting 18S rDNA. The assay was examined for cross reactivity by testing additional Thaumatotibia species and representatives of related Olethreutine leafroller genera such as Cryptophlebia, Cydia and Grapholita, which are often encountered in the same geographic region and on the same hosts as T. leucotreta. We compared our newly developed test to previously published TaqMan real-time PCR, SYBR Green real-time PCR and loop-mediated isothermal amplification (LAMP) tests. Our newly developed real-time PCR assay outperformed all three tests in terms of analytical specificity with 100% accuracy. These results will help to further improve diagnostic standards for molecular identification of T. leucotreta.